The level of free intracellular methotrexate and the capacity for polyglutamation of methotrexate may determine the duration of tetrahydrofolate synthesis. The relationship between methotrexate polyglutamate synthesis as a function of free or exchangeable methotrexate will be studied in the Ehrlich ascites tumor and liver cells. The possible inhibition of mammalian thymidylate synthetase from the accumulation of intracellular methotrexate-polyglutamate will be studied. An evaluation of methotrexate-polyglutamates as a determinant of methotrexate toxicity will be determined in mice treated with various combinations of methotrexate, vincristine, and thymidine. An analysis of the membrane transport characteristics of methotrexate at extracellular concentrations relevant to drug levels achieved in high-dose methotrexate regimens in the presence and absence of vincristine will be investigated. The role of fluoropyrimidines and methotrexate transport will be examined to aid in defining the proper sequence and interval between administration of these agents so that synergistic interactions are realized as a result of biochemical changes they produce.